Understanding connectional deficits in mutant and transgenic mouse models is essential to a mechanistic understanding of how such deficits contribute to nervous and sensory system disorders. To achieve this requires neurotracing reagents that a) can delineate nerve fibers even in partially defunct cells and b) allow simultaneous tracing of multiple nerve tracts in embryos, juveniles and adults, with equivalent resolution for thick vs. thin and myelinated vs. unmyelinated fibers. Because all nerve cells have membranes, even when defects in critical genes cause aberrant development, lipophilic fluorescent dyes can provide information where other techniques may fail. Tracing with such dyes has been limited by time consuming and cumbersome methodology, largely due to poorly matched spectral and diffusional properties. The project?s long term goal is to build a family of lipophilic fluorescent dyes/reporter molecules to maximize the number of neuronal tracts which can be traced at the same time in individual fixed and/or live specimens. In Phase I, a novel set of lipophilic NeuroVue? dyes with well matched spectral and diffusion properties were developed and commercialized in a simple-to-use coated filter format. These new products enable up to 3- color studies on standard confocal systems and up to 5 color studies on enhanced systems with spectral detection/color unmixing capabilities. This SBIR Phase II proposal will add violet, near-infrared and time- resolved NeuroVue dyes to further increase the number of fibers that can be traced simultaneously, and creation of specialized probe sets for i) detection of developing neuronal connections and ii) long-term neurotracing studies in myelinated/adult tissue. Phase II specific aims are to:1) Expand the Phase I NeuroVue Standard dye set to enable simultaneous tracing of up to 5 neuronal tracts in tissues requiring diffusion periods of at least 4 weeks using standard confocal microscopy; 2) Create optimized dye pair enabling detection of neuronal proximity/connection formation in fixed tissue using standard confocal microscopes (NeuroVue Connections); 3) Expand the Phase I NeuroVue Enhanced set to enable simultaneous tracing of at least 8 neuronal tracts over a diffusion period of at least 4 weeks using enhanced confocal systems with spectral detection/color unmixing, 2-photon excitation and time-resolved fluorescence detection capabilities; 4) Create a dye set optimized for long-term (at least 6 week diffusion) studies of myelinated fibers (e.g. ,tissues from adult mice and, potentially, fixed human specimens), 5) Commercialize the best new dyes from Specific Aims 1-4. Completion of the workplan proposed will: a) Provide a family of well-matched neurotracing probes enabling at least 5-color neurotracing on standard confocal systems and 8-color studies on advanced confocal systems; b) Significantly reduce the cost and number of animals required to trace neuronal circuits, and c) Provide novel tools to study how development of cellular connections are affected by mutations, and are remodeled in the presence of disease, damage or aging. This work will provide novel tools for the study of how connections in the nervous system are altered by genetic defects, disease, trauma or aging. The new techniques will allow more information to be gained from fewer animals, leveraging existing NIH investments in the development of thousands of mutant mouse models with defined genetic defects. These tools and models are expected to provide the neuroscience community with critical insights into the developing brain and how to design more effective therapeutic interventions. [unreadable] [unreadable] [unreadable]